Synthesis and evaluation of 4,5-dihydro-5-methylisoxazolin-5-carboxamide derivatives as VLA-4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.12.043

A novel set of compounds containing a 4,5-dihydro-5-methylisoxazoline have been successfully designed as VLA-4 receptor antagonists. Compound (14p) had a high receptor binding affinity of 4 nM and also found to be metabolically stable in vitro.

Full text of DOI:10.1016/j.bmcl.2012.12.043

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1482–1485
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and evaluation of 4,5-dihydro-5-methylisoxazolin-5-
carboxamide derivatives as VLA-4 antagonists
a
a
b
c
b
Ajay Soni a, , Abdul Rehman , Keshav Naik , Sunanda Dastidar , M. S. Alam , Abhijit Ray ,
⇑
Tridib Chaira b, Vanya Shah b, Venkata P. Palle a, Ian A. Cliffe a, Viswajanani J. Sattigeri a
a Department of Medicinal Chemistry, New Drug Discovery Research, Ranbaxy Research Laboratories, Plot 20, Sector 18, Gurgaon 122015, Haryana, India
b Department of Pharmacology, New Drug Discovery Research, Ranbaxy Research Laboratories, Plot 20, Sector 18, Gurgaon 122015, Haryana, India
c Department of Chemistry, Jamia Hamdard, New Delhi 110062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel set of compounds containing a 4,5-dihydro-5-methylisoxazoline have been successfully designed
as VLA-4 receptor antagonists. Compound (14p) had a high receptor binding affinity of 4 nM and also
found to be metabolically stable in vitro.
Received 30 August 2012
Revised 12 December 2012
Accepted 14 December 2012
Available online 21 December 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
VLA-4
Proline
Isoxazoline
Antagonist
Very late antigen-4 (VLA-4,
a
4b1) is a member of the b1 sub-
The synthesis of 3-substituted isoxazolines began with the
reaction of aldehydes (8a–8t) with hydroxylamine to give the cor-
responding oxime derivatives (9a–9t) in moderate to good yields
(Scheme 1). The oximes were cyclized with methyl methacrylate
under oxidizing conditions to produce the isoxazolines (10a–10t)
which were hydrolyzed under basic conditions to give the corre-
sponding acids (11a–11t). The acids were coupled with the phen-
ylalanine derivative (12)16 and the resulting products (13a–13t)
were hydrolyzed to produce the desired acids (14a–14t) in quanti-
tative yields. The tyrosine derivatives (15a)15 and (15b)17 were
coupled with isoxazoline (11a) to produce the coupled products
(16a, 16b) which on hydrolysis gave compounds (17a, 17b)
(Scheme 2).
The compounds were evaluated for their ability to inhibit the
binding of VLA-4 to VCAM-1 in a U-937 (human T cell line) cell
adhesion assay (Tables 1and 2).18 The data on the 3-arylisoxazoline
series (14a–14i) revealed that, apart from the 3-chloro substituted
compound (14a), the unsubstituted compound (14b) displayed the
most potent VLA-4 inhibition activity [compare compound (14b)
with compounds containing 2-chloro (14c), 2-fluoro (14d),
4-methyl (14e) and 2-methoxy (14f)]. It was also observed
that mono-substituted compounds were more potent than di-
substituted compounds [compare compounds 14a, 14c–14f with
compounds 14g–14i)]. The high activity of the 3-chloro compound
(14a) and the low activity of the 3,4-dimethoxy compound (14g) is
difficult to interpret unless there exists perhaps a favorable steric
interaction at the meta-position which is unfavorable at the para-
position.
family of integrins and is expressed on all circulating leukocytes
apart from platelets.1,2 VLA-4 mediates both cell–cell and cell–
matrix interactions by binding to cell surface ligands such as
vascular cell adhesion molecule-1 (VCAM-1) and cell–matrix pro-
teins such as fibronectin. These interactions are important for the
activation, migration and proliferation of leukocytes during normal
and pathophysiological processes, and so the inhibition of VLA-4 is
expected to be of therapeutic benefit in the treatment of a variety
of inflammatory diseases3,4 such as asthma, multiple sclerosis, and
rheumatoid arthritis.
VLA-4 binds to
a protein motif containing the sequence
EILDVPST within VCAM-1, and peptidomimetic inhibitors which
bind to the critical leucine-aspartic acid-valine (LDV) sequence
have been reported.5 Non-peptidomimetic inhibitors have also
been developed (Fig. 1) in which an N-acylphenylalanine core ap-
pears to be critical for activity.6–14 As a part of our ongoing re-
search, we sought to obtain another suitable replacement for the
N-acyl group in compounds (1–5).
Previously, we reported the evaluation of 1,3-dioxolanes (6)15
as proline bioisostere. Herein, we report the synthesis and evalua-
tion of compounds (7) containing a 4,5-dihydro-5-methylisoxazo-
lin-5-carboxamide group as yet another successful replacement for
the proline/thiaproline group in standard VLA-4 inhibitors (Fig. 2).
⇑
Corresponding author.
E-mail address: ajay_soni_78@yahoo.com (A. Soni).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.043

A. Soni et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1482–1485
1483
H
N
H
N
H
N
N
S
N
S
OH
OH
O
O
O
O
O
O
O
O
O
N
H
(1) American Home Product/Elan CT-757
(2) American Home Product/Elan
H
H
N
N
H
S
Cl
N
N
S
O
OH
O
N
O
O
O
Cl
N
OH
O
O
O
O
Cl
Cl
(4) Celltech, CT-5219
(3) Merck
O
H
N
O
OH
(5) Merck
Figure 1. Potent VLA-4 antagonists.
O
O
O
R2
R
R2
R
O
(i)
OH
O
O
O
N
(11a)
+
O
O
N
N
H
N
H
O
O
OH
O
N
O
H2N
O
O
H
O
O
R1
HN
R1
(6)
(16a,16b)
(7)
(15a) R = 2,6-(OMe)2-C6H3
(ii)
Figure 2. 1,3-Dioxolane-based and isoxazoline-based VLA-4 inhibitors.
(15b) R = (2,6-Cl2-C6H3)CH2O
R
O
(iv)
OH
H
(ii)
O
N
(i)
N
O
R1
R2
R
H
R
(12)
O
O
OH
N
N
H
O
(8a-8t)
(9a-9t)
O
(10a-10t) R1 = Me
(11a-11t) R1 = H
(iii)
(17a,17b)
Scheme 2. Reagents and conditions: (i) N-methylmorpholine, HOBt, EDC, DMF,
50–55%; (ii) LiOH, THF, MeOH, H2O, 80–90%.
Cl
Cl
H
N
H
N
(v)
O
Cl
O
Cl
of 4 nM. This high potency may arise from either a simple steric
effect or more likely an electron donating effect which increases
the negative charge on the isoxazoline ring nitrogen atom. The 3-
cyclohexyl derivative (14q) had higher affinity than the 3-phenyl
derivative (14b) which implies that the larger steric size or the
positive inductive effect of the cyclohexyl group is more important
than the smaller steric bulk or the positive mesomeric effect of the
phenyl ring. The 3-methyl derivative (14r) had weaker activity
than the tert-butyl and cyclohexyl derivatives (14p) and (14q) as
O
O
N
O
OH
N
N
N
H
H
O
O
R2
R2
O
O
(14a-14t)
(13a-13t)
Scheme 1. Reagents and conditions: (i) hydroxylamine hydrochloride, sodium
acetate, ethanol, rt, 50–90%; (ii) methyl methylacrylate, sodium hypochlorite, THF,
rt, 60–80%; (iii) LiOH, THF, MeOH, H2O, 75–80%; (iv) methyl 4-[(2,6-
dichlorobenzoyl)amino]-L-phenylalaninate (12), N-methylmorpholine, HOBt, EDC,
a
result perhaps of reduced size or inductive effects. The
DMF, 80–90%; (v) LiOH, THF, MeOH, H2O, 100%.
3-(1-phenylethyl) and 3-(2-phenylethyl) derivatives (14s and
14t) had weak activity and may be encumbered sterically.
The 3-heteroarylisoxazolines (14j–14o) displayed a range of
activities although in general the ring-fused bi-aryl systems
(14j–14l) showed higher inhibitory activity than the mono-aryl sy-
tems (14m–14o). There exists in these cases a complex interplay of
steric and electronic forces and no correlation exists between bio-
logical activity and isoxazoline electron density. The most
potent compounds were found among the 3-alkylisoxazolines
(14p–14r). The 3-(tert-butyl) derivative (14p) had an IC50 value
Finally, an investigation of the effect on binding to the VCAM-1
receptor of the presence of a substituent placed at the para-
position of the phenyl ring of the phenylalanine unit was under-
taken (Table 2). Both the 2,6-dimethoxyphenyl (17a) and the 2,6-
dichlorophenylmethyl ether (17b) derivatives were found to have
lower binding affinities than the 2,6-dichlorophenylcarboxamide
(14b). The threefold lower affinity of compound (17b) compared
with compound (14b) is in line with earlier work15 in which an

1484
A. Soni et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1482–1485
Table 1
Binding affinities of compounds (14a–14t)
Cl
H
N
O
Cl
O
O
OH
N
N
H
O
R
Compd
R
IC50
(
lM)
n
Compd
R
IC50
(
lM)
n
Cl
14a
0.29 0.07
0.69 0.05
3
3
14b
0.37 0.02
1.20
3
Cl
F
14c
14e
14d
14f
1
1
MeO
Me
1.20
1.90
1
1
1.80
3.5
MeO
MeO
OMe
OMe
14g
14h
1
Cl
N
14i
4.80
0.76
1
2
14j
14l
0.43
0.98
2
2
Cl
N
14k
S
S
14m
14o
0.89
2.90
2
1
14n
14p
1.40
2
3
N
N
0.004 0.002
14q
14s
0.16 0.05
3
1
14r
14t
–Me
0.83
2.70
2
1
Me
(CH2)2
1.50
analogous substitution pattern in compound (6) gave derivatives
having a fivefold separation in activity. The presence of a steric
interaction at the para-position of the phenylalanine was the rea-
son given previously for this effect and a similar explanation may
be offered here.
An overlay of the low energy structures of compound (14p)
with CT-5219 (4) reveals that the molecules adopt similar confor-
mations with key functional groups and heteroatoms being located
in similar positions: thus, the N-acylphenylalaninyl units are over-
lapped, and the isoxazoline ring of compound (14p) points in a
similar direction to the 3-acetylthiazolidine in compound (4)
(Fig. 3).
encouraged to see that all selected compounds were stable in hu-
man, rat and mouse microsomes (Table 4). Further, the compound
14p did not exhibit any liability to inhibit the major CYPs up to
10 lM concentration (Table 3). Compound 14p was also taken up
in a rat PK study. Intravenous pharmacokinetic study of 14p
in Sprague–Dawley rat showed moderate plasma clearance
(28.61 mL/min/kg), low distribution in tissues (0.53 L/kg), short
terminal half-life of 1.4 h and poor bioavailability. The low volume
of distribution suggests less potential for organ toxicity (Table 5).
Additionally, compound 14p was also profiled in rat S9 and
showed excellent stability (91.6% remaining in 30 min). The reason
for the observed poor bioavailability may need further investiga-
tion, and may be the subject of future communication.
Historically, the VLA-4 program has been fraught with poor
pharmacokinetic profiles of potent inhibitors. Hence, to check the
pharmacokinetic profile of isoxazoline compounds (14p–14t),
ADME profile of a few 3-alkylisoxazolines was assessed. We were
In summary 4,5-dihydro-5-methylisoxazolin-5-carboxamide
group was identified as a viable replacement of the N-acyl group
present in compounds (1–5). The 3-(tert-butyl)-4,5-dihydo-5-

A. Soni et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1482–1485
1485
Table 2
Table 5
Binding affinities of compounds (14b, 17a, 17b)
Pharmacokinetic properties of compound (14p) in rats
Cmax
(l
g h/mL)
Tmax (h)
AUC0–inf
(lg h/mL)
F(%)
R
po (10 mg/kg)
0.07
0.25
0.24
4
O
O
C0
(l
g/mL)
AUCinf
2.33
(l
g h/mL)
T1/2 (h)
CL (mg/min/kg)
28.61
Vss (L/kg)
OH
N
N
H
iv (4 mg/kg)
12.42
O
1.4
0.53
(0.53 L/kg), short terminal half-life of 1.4 h and poor bioavailability
(4%). Further optimization of these lead compounds with improved
PK profile is underway and will be the subject of future
publications.
Compd
R
IC50
(
l
M)
n
Cl
H
N
14b
0.37 0.02
2.70
3
O
Cl
OMe
Cl
Acknowledgments
MeO
17a
17b
1
3
We thank Dr. Jitendra A. Sattigeri for his kind guidance and the
Analytical Chemistry Department of the New Drug Discovery
Research Division for providing spectral data.
Cl
O
1.20 0.15
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Figure 3. Structural overlay of CT-5219 (Purple) and 14p (Green).
Table 3
% CYP inhibition (at 10
l
M) compound (14p)
1A2
21
2C9
7
2C19
22
2D6
6
3A4
5
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Compd
Human
Mouse
Rat
14p
14q
14r
14s
14t
98
93
100
95
96
100
100
99
96.5
77.7
116.6
84.5
—
65
100
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inhibition of VLA-4 binding to the VCAM-1 receptor with an IC50
value of 4 nM. 3-Alkylisoxazolines were found to be stable in
human, rat and mouse microsomes. Further, pharmacokinetic
study (iv, 4 mpk) of 14p in Sprague–Dawley rat showed moderate
plasma clearance (28.61 mL/min/kg), low distribution in tissues
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